The fluidic subsystem is, perhaps, the easiest of the three hardware subsytems to understand. In essence, it consists of a sheath/buffer fluid source, the sample source itself, a flow cell and a waste collection system.
As you may recall, the sheath fluid and sample fluid are injected into the flow cell from their respective reservoirs in order to produce hydrodynamic focusing and smooth laminar flow directing the cells, single file, through the flow cell. This injection can be achieved in any number of ways, but a classic cytometer will use air pressure and a differential control system.
In a differential control system, air is pumped into the sheath reservoir at a controlled pressure (let’s say 13psi). When the fluidic lines are open, this pressure will push the sheath through the system at a steady rate. In order to move the sample fluid, the air source is split and the sample pressure line is directed through a biasing relay or differential valve. This differential will reduce the air pressure by a small fraction before pressurizing the sample chamber. (Note: the fluid dynamics of the flow cell allows the sample injection to occur even when the sample pressure is lower than the surrounding sheath pressure). Controlling the sample flow rate, then, is simply a matter of controlling the amount of pressure reduction over the sample. Too much reduction will, of course, result in loss of sample flow and, in extreme cases, back flow of the sheath fluid into the sample lines. Too little reduction will result in an expanded center stream with poor focusing characteristics.
Typical Flow Cells
This image shows a typical closed, or cuvette, flow cell. It is, in fact, the flow cell from a Beckman-Coulter FC-500 desktop analyzer. As you can see hydrodynamic focusing occurs within the conical section of the flow cell. However, in this case, the fluid stream is not released in free space. Rather, the flow cell extends into an optical cuvette, and it is within this cuvette that inspection of the cell occurs. This cuvette is designed to minimize loss of excitation and emission photons and to direct the emitted light to the collection optics. As can be expected, these systems provide improved sensitivity over stream-in-air systems, but are not well-suited to the droplet formation required for electrostatic cell sorting (with, perhaps, the notable exception of the flow cell as utilized in the BDBiosciences FACSAria Cell Sorter).
The Final Step After inspection, the cells are directed to their final destination. In an analyzer, the entire stream is sent to a waste tank for disposal. In a cell sorter, droplets containing the desired cells are diverted to an appropriate collection receptacle with the remainder of the the stream fluid being diverted to a waste tank for disposal. In the earliest cell sorters, this waste tank was often a beaker or flask sitting beneath the flow cell!
Moving On Once you’re comfortable with this material, please move on to the next subsystem.